Synopsis Cells contain an essentially continuous network of organized molecules, with large structures such as cytoskeletal “girders” and membrane domains forming a shifting scaffold for the organization of many smaller molecular assemblies. These structures are highly dynamic; both their position and composition are controlled to regulate cell behavior and integrate information traveling through different signaling pathways. Such dynamic supramolecular organization is difficult to understand by examining fixed cells or isolated proteins in vitro. We are developing new tools to visualize and quantify protein behavior within living cells, and to reversibly modulate protein activities in specific subcellular locations. Our work involves close collaboration between organic chemists and ‘protein engineers’, working with molecular biologists and cell biologists to shed light on signaling. We are focusing on a signaling network that controls many different, sometimes opposite, cell behaviors. GTPases, MAP kinases and adhesion molecules rely on the formation of localized complexes and scaffold interactions to control the flow of information regulating neuronal development, cell movement, and engulfment behaviors such as phagocytosis and macropinocytosis. This ubiquitous signaling network controls fundamental behaviors important in cancer, cardiovascular disease, and other pathologies. We use biosensors to characterize the localized activation of signaling proteins in these pathways, then modulate upstream regulatory molecules using traditional approaches and novel methods for light-mediated manipulation of signaling activity with precise kinetics. Through genome-wide screens based on robotic microscopy we are identifying new pathways. Collaborations with colleagues modeling biosensor behavior are enabling us to produce far more quantitative and less perturbing measurements from within cells, ultimately to provide data for larger scale models of cellular signaling networks. We hope our work will be valuable both for shedding light on previously inaccessible mechanisms of signaling control, and by providing broadly applicable and easy to use tools.